Academic journal article Genetics

The Dominant Inhibitory Chalcone Synthase Allele C2-Idf (Inhibitor Diffuse) from Zea Mays (L.) Acts Via an Endogenous RNA Silencing Mechanism

Academic journal article Genetics

The Dominant Inhibitory Chalcone Synthase Allele C2-Idf (Inhibitor Diffuse) from Zea Mays (L.) Acts Via an Endogenous RNA Silencing Mechanism

Article excerpt


The flavonoid pigment pathway in plants has been used as a model system for studying gene regulatory mechanisms. C2-Idf is a stable dominant mutation of the chalcone synthase gene, c2, which encodes the first dedicated enzyme in this biosynthetic pathway of maize. Homozygous C2-Idf plants show no pigmentation. This allele also inhibits expression of functional C2 alleles in heterozygotes, producing a less pigmented condition instead of the normal deeply pigmented phenotype. To explore the nature of this effect, the C2-Idf allele was cloned. The gene structure of the C2-Idf haplotype differs substantially from that of the normal c2 gene in that three copies are present. Two of these are located in close proximity to each other in a head-to-head orientation and the third is closely linked. Previous experiments showed that the lower level of pigmentation in heterozygotes is correlated with reduced enzyme activity and low steady-state mRNA levels. We found that c2 transcription occurs in nuclei of C2-Idf/C2 heterozygotes, but mRNA does not accumulate, suggesting that the inhibition is mediated by RNA silencing. Infection of C2-Idf/C2 heterozygotes with viruses that carry suppressors of RNA silencing relieved the phenotypic inhibition, restoring pigment production and mRNA levels. Finally, we detected small interfering RNAs (siRNAs) in plants carrying C2-Idf, but not in plants homozygous for the wild-type C2 allele. Together, our results indicate that the inhibitory effect of C2-Idf occurs through RNA silencing.

THE maize flavonoid pigment pathway offers an excellent model system for studying the regulation of gene expression. The pathway is genetically well characterized and most of the structural and regulatory genes have been cloned (DoONER et al. 1991). Genetic and molecular studies of a wide spectrum of mutants have revealed that the distribution and level of pigment accumulation accurately reflect activity of these genes (CoE et al. 1988). In maize, several genes involved in anthocyanin biosynthesis have been identified. Among these, regulatory genes encoding a suite of transcription factors as well as structural genes encoding biosynthetic enzymes have been extensively characterized at the molecular level (DoONER et al. 1991). From these data, detailed knowledge about the regulation, mode of interaction, and function of maize anthocyanin genes has been assembled.

One of the structural genes, colorless2 (c2), encodes chalcone synthase, the enzyme responsible for the first dedicated step in the pathway. In combination with appropriate regulatory alleles, a normal C2 allele leads to pigment production in many parts of the plant, including the pericarp, the aleurone layer of the endosperm, tassels, and vegetative organs such as ear husks and leaf sheaths. A colorless mutant, initially called Inhibitor diffuse (Idf), was isolated from Peruvian lines as a dominant inhibitor of pericarp pigmentation (BRINK and GREENBLATT 1954). Later, when the mutation was mapped to the c2 locus, the allele became known as C2/^/(BRINK and GREENBLATT 1954). When heterozygous with a normal C2 allele, C2-Idf reduces pigmentation not only in the pericarp, but also in the aleurone. At the enzyme level, the effect on aleurone pigmentation is due to a lack of chalcone synthase enzyme activity (DOONER 1983). At the RNA level, c2-homologous RNA is not detectable in either tassels or aleurone of C2-Idf homozygotes (FRANKEN et al. 1991).

Unlike cases of paramutation (for reviews see CHANDLER and STAM 2004; BELLA VEDOVA and CONE 2004), C2-/i^inhibition is not meiotically heritable. These observations suggest that the absence of enzyme activity in C2-Idf/C2 hétérozygotes results from a reduction of C2 mRNA accumulation. In this work, we investigated the mechanism of inhibition of the C2 allele by C2-Idf.

Similar semidominant types of mutations in chalcone synthase genes have been described and analyzed in Antirrhinum majus L. …

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